Impact of Behavioral Assessment and Re-Test as Functional Trainings That Modify Survival, Anxiety and Functional Profile (Physical Endurance and Motor Learning) of Old Male and Female 3xTg-AD Mice and NTg Mice with Normal Aging

Longitudinal approaches for disease-monitoring in old animals face survival and frailty limitations, but also assessment and re-test bias on genotype and sex effects. The present work investigated these effects on 56 variables for behavior, functional profile, and biological status of male and female 3xTg-AD mice and NTg counterparts using two designs: (1) a longitudinal design: naïve 12-month-old mice re-tested four months later; and (2) a cross-sectional design: naïve 16-month-old mice compared to those re-tested. The results confirmed the impact as (1) improvement of survival (NTg rested females), variability of gait (3xTg-AD 16-month-old re-tested and naïve females), physical endurance (3xTg-AD re-tested females), motor learning (3xTg-AD and NTg 16-month-old re-tested females), and geotaxis (3xTg-AD naïve 16-month-old males); but (2) worse anxiety (3xTg-AD 16-month-old re-tested males), HPA axis (3xTg-AD 16-month-old re-tested and naïve females) and sarcopenia (3xTg-AD 16-month-old naïve females). Males showed more functional correlations than females. The functional profile, biological status, and their correlation are discussed as relevant elements for AD-pathology. Therefore, repetition of behavioral batteries could be considered training by itself, with some variables sensitive to genotype, sex, and re-test. In the AD-genotype, females achieved the best performance in physical endurance and motor learning, while males showed a deterioration in most studied variables.


Introduction
Specific motor skills impaired in old age include a broad and varied spectrum that involves a reduction in gait speed, loss of strength and muscle mass, and decline of balance [1][2][3]. However, aging has become increasingly recognized as a potentially modifiable risk factor for chronic disease and frailty [4,5]. The deterioration of motor performance related to cognitive dysfunction in Alzheimer's disease (AD) has recently gained importance in clinical research [6][7][8][9]. Particularly, gait impairment and its association with cognitive impairment [10] could shed light on potentialities to distinguish AD [1]. Inclusive, higher levels of Aβ and tau are associated with more significant memory decline, but not with changes in executive function [11]. The study by Sperling points out that these results could explain why some clinically active patients presented elevated tau and Aβ levels [11]. Thus, Aβ and tau proteins can serve as markers of cognitive impairment; however, they are insufficient and cannot detect all cases of dementia, especially in the early stages [11,12]. For this part, gait speed, for example, is longitudinally associated with cognitive decline, Figure 1. Experimental design. Longitudinal design: the first group was assessed in the behavioral battery at the age of 12 months and again when the animals reached 16 months of age. Transversal design: the second group was housed in standard conditions without manipulation until they were tested at 16 months of age, so they could be compared to re-tested 16 months old animals.

Behavioral Assessment and Biological Status
The assessment consisted of four consecutive evaluation steps conducted during 5 days, as follows: Day 1, bodyweight, phenotype scoring system, and frailty; Day 2, gait and exploration; Day 3, geotaxis, muscle strength, and rotarod; Days 4-5, rotarod. The procedures and protocol were based on the protocol used by Castillo-Mariqueo and Giménez-Llort [31]. Assessments were performed under dim white light (20 lx) in the light cycle (10:00 a.m. to 1:00 p.m.). Behavioral evaluations were carried out in a counterbalanced way by two independent observers, blind to the genotype. Animals were habituated to the test room 30 min before the start of the tests.
2.3.1. Survival, Bodyweight, Phenotype Scoring System, Frailty Score, and Kyphosis Survival curves were analyzed considering the cohort of siblings from the same litter of mice included in the study, from birth to 16 months of age. A total of 191 male and female mice, NTg and 3xTg-AD were included in this analysis (NTg males = 49; NTg fe- Figure 1. Experimental design. Longitudinal design: the first group was assessed in the behavioral battery at the age of 12 months and again when the animals reached 16 months of age. Transversal design: the second group was housed in standard conditions without manipulation until they were tested at 16 months of age, so they could be compared to re-tested 16 months old animals.

Quantitative Parameters of Gait, Neophobia, and Exploration
The quantitative parameters of the gait and exploration were recorded by filming the spontaneous gait of the mice for 1 min. Later the videos were analyzed using KINOVEA 0.8.15 free software according to the Castillo-Mariqueo and Gimenez-Llort protocol [31]. Stride length, stride length variability, speed, and cadence were included according to the methodology used by Wang et al. [43]. The examination included observation of body position, limb support, and movement. In addition, neophobia (immediate fear of a new place) was assessed by means of the corner test [44] and the recording of freezing (latency of movement), the number of explorations on the horizontal axis (visited corners), the latency and number of explorations on the vertical axis (rearings).

Muscular Strength-Hanger Test and Geotaxis
The muscle strength was measured in the forelimbs using the hanger test. Three trials were performed to observe the tendency of a mouse to instinctively grasp a rack or bar when suspended by the tail. In the first and second trials, grip strength was assessed by holding the animal with its front legs for 5 s at the height of 40 cm. In the third trial, the animal is suspended for 60 s in a single attempt to assess muscular endurance. This test allows discriminating grip strength and muscular endurance according to the suspension times used by mice [45]. A box with sawdust is placed under the animal to prevent a possible fall in each trial. The bar used is graduated in 5-cm blocks to obtain the distance covered when the animal moves through the bar. The latency and movement distance are recorded. Geotaxis was measured using a 10 cm×12cm grid. A single trial registered the time it took for the animal to reach the vertical position from an inverted position at a 90 • angle on the grid.

Motor Performance: Learning and Physical Endurance-Rotarod
Six micro training cycles were carried out during three consecutive days with a previous learning session and psychomotor coordination. The animals were trained in the Rotarod apparatus (Ugo basile ® , Mouse RotaRod NG) according to a training volume established in our previous research laboratory investigations [31]. An incremental intensity of 5 to 48 rpm was applied according to individual tolerance with a maximum duration of 360 s in each microcycle with a 1-min recovery between trial.

Biological Status: HPA Axis and Sarcopenia Index
The animals were euthanized and the muscle tissues were necropsied. Plasma from a blood sample was obtained by centrifugation and stored and −80 • C until corticosterone analysis. Corticosterone content (ng/mL) was analyzed using a commercial kit (Corticosterone EIA Immunodiagnostic Systems Ltd., Boldon, UK). Absorbance was read at 450 nm with Varioskan LUX ESW 1.00.38 (Thermo Fisher Scientific, Massachusetts, MA, USA) [42]. The weights of the quadriceps and triceps surae muscles of the right lower extremity of each animal were recorded and kept for future analysis. The sarcopenia index [46] was applied to obtain an indirect measure of sarcopenia as a biological marker of frailty.

Statistics
Statistical analyses were performed using SPSS 15.0 software. Results were expressed as the mean ± standard error of the mean (SEM) for each task and trial. The variables recorded were analyzed with Student t-test, Chi-squared or Fisher's exact test, one-way ANOVA, and multiple regression analysis (MRA). The split-plot ANOVA design with factors genotype (G), sex (S), previous experience either as a re-test (R) in the longitudinal approach or as naïve (N) in the transversal approach, were included. Their G×S, G×R, and S×N factor interactions were also studied. Post hoc comparisons were run with Bonferroni corrections. Pearson's correlations were made to analyze the functional correlations with (1) corticosterone, (2) sarcopenia index, and (3) phenotype score system. The survival curve was analyzed with the Kaplan-Meier test (Log rank). In all cases, p < 0.05 was considered statistically significant.
Biomedicines 2022, 10, 973 8 of 28 In bodyweight at 12 months was high in 3xTg-AD, and at 16 months it decreased in females. In addition, males naïve 16 months weighed more than re-test males at the same, see Tables S1-S3.

Quantitative Parameters of Gait, and Neophobia and Exploration
Quantitative parameters of gait are shown in Figure 3. For naïve 12 months, statistically significant differences were observed in all quantitative gait variables. Stride length showed differences in G and S, with the longest stride length in NTg males and 3xTg-AD in females. This interaction was also observed in gait speed, high in NTg mice. At the same time, the variability of gait presented differences associated with S, with females showing less than males' variability and, therefore a gait with more homogeneous steps in its trajectory. Additionally, a genotype-dependent difference was observed in cadence, where NTg mice show better performance in this variable with marked differences between males. In the re-test at 16 months, this group registered a gait performance that shows the interaction between the G×S effect in stride length and speed, with the performance of 3xTg-AD females being the one with the best performance in both variables. The re-test of this group at 16 months showed differences in cadence, increasing its performance in the group of 3xTg-AD mice of both sexes and decreasing in the NTg group, see Figure 3A-D and Tables S1-S3.
On the other hand, mice at 16 months did not show significant differences in quantitative variables of gait. Differences could only be observed between the re-test and a naïve group of males at 16 months in stride length, cadence, and speed, where the re-test NTg mice presented a high performance in speed and cadence compared to the naïve NTg mice, 3xTg-AD re-test, and naïve, but lower performance in stride length than naïve mice. In addition, differences were detected between the 3xTg-AD males and females in the variable's variability and gait speed, with the 3xTg-AD naïve and re-test females showing less variability than the 3xTg-AD males. This difference was also present in gait speed, with a better performance of re-test females followed by naïve 16 months females over 3xTg-AD males in both conditions, see Figure 3A-D and Tables S1-S3.
For its part, the neophobia and exploratory activity presented sex differences in the ratio visited corners/rearings, being higher in females of both genotypes, see Figure 4. This difference was maintained at re-test 16 months, with the higher ratio in females. In addition, the ratio in MRA of the groups showed an interaction between G×S, indicating a lower performance in 3xTg-AD re-test males at 16 months and higher in re-test females, see Figure 4B and Tables S1-S3.
As in gait, no significant differences were detected in exploratory activity between the naïve 16 months group. However, in contrast to the re-test males at 16 months, the naïve males presented high vertical activity than the re-test, see Figure 4C,D. Between the group of 3xTg-AD mice, S effect was identified in vertical activity, where naïve males presented higher activity. Movement latency was also lower in naïve males, but the same was not observed in 3xTg-AD females, see Figure 4A and Tables S1-S3.

Muscular Strength: Forelimb Grip Strength and Muscular Endurance-Hanger Test and Response to Gravity: Geotaxis
Lower muscle strength can be observed in the resistance distance in the 3xTg-AD animals at the age of 12 months, which, despite not showing statistical differences, shows a trend with less strength in the 3xTg-AD males. No statistically significant differences were detected in the rest of the variables, although a worse performance of the animals was observed, see Table S1. At 16 months re-test and 16 months naïve, no significant differences were detected.
On the other hand, significant differences in geotaxis were detected. The group of 3xTg-AD male took longer to complete the test in the re-test 16 months group in contrast to the 12 months and naïve 16 months group, see Figure 5A. Notably, at 16 months in the re-test group, an interaction was observed between the G×S of the animals, showing more significant latency in 3xTg-AD males and NTg females. G×S interaction was also observed between the group of naïve 16 months 3xTg-AD mice. In addition, there was an interaction between the G×R was detected among male mice at 16 months in contrast to naïve mice of the same age, with the test time being shorter in naïve mice, see Figure 5A and Tables S1-S3. cadence. Statistics: ANOVA, G, genotype effect, G** p < 0.01**, G* p < 0.05*. S, sex effect, S* p < 0.05*. G×S, genotype and sex interaction effects, G×S* p < 0.05*. R, re-test effect, R* p < 0.05*. N, naïve effect, N* p < 0.05*. a, aging, a** p < 0.01**. Bonferroni post hoc test: g, genotype; s, sex; $ expressed genotype differences between sex, and # expressed sex differences between genotypes. The symbol ⦸ indicates the absence of the group, and m, month. cadence. Statistics: ANOVA, G, genotype effect, G** p < 0.01**, G* p < 0.05*. S, sex effect, S* p < 0.05*. G×S, genotype and sex interaction effects, G×S* p < 0.05*. R, re-test effect, R* p < 0.05*. N, naïve effect, N* p < 0.05*. a, aging, a** p < 0.01**. Bonferroni post hoc test: g, genotype; s, sex; $ expressed genotype differences between sex, and # expressed sex differences between genotypes. The symbol    Statistics: ANOVA, G, genotype effect, G* p < 0.05. S, sex effect, S** p < 0.01, S* p < 0.05. R, Retest effect, naïve 12 months and re-test 16 months, R** p < 0.01**, R* p < 0.05*. G×S, genotype and sex interaction effects, G×S** p < 0.01**, G×S* p < 0.05*. G×R, genotype and re-test effects, G×R** p < 0.01**, G×R* p < 0.05*. Bonferroni post hoc test: g, genotype, s, sex, r: re-test naïve 12 months vs. 16

Motor Performance: Learning and Physical Endurance-Rotarod
The learning and motor performance tests in the Rotarod showed significant differences associated with different factors depending on the test or the group studied, see Figure 5. Among the males, significant differences were detected in learning and the number of trials between naïve and re-tests at 12 months and 16 months. In females, differences were detected in 3xTg-AD of 12 months and 16 months re-test and naïve, see Figure 5B,C and Tables S1-S3. In turn, for motor learning, the S effect plays an important role since females manage to learn earlier than males and spend more time on the wheel during the test at 12 months. At the re-test 16 months, the S effect was maintained in the number of trials, but in learning the G effect and G×S became important. In the same way, when performing MRA in the groups naïve at 12 months and re-test at 16 months, the S effect was the one that marked the statistical difference, see Figure 5B,C and Tables S1-S3. Nevertheless, there were no significant differences between the naïve 16 months group. However, between the re-test 16 months group vs. the naïve 16 months, differences were detected between males, where the R and G effects were significant. In addition, significant differences were also detected between the 3xTg-AD group, where the differences in S and R effects were the ones that obtained significance, see Figure 5B,C and Tables S1-S3.
At the same time, it is possible to differentiate physical endurance according to the interaction of G and S in the naïve 12 months, re-test 16 months, and naïve 16 months groups, see Figure 6. The NTg males have a physical endurance similar to that of 3xTg-AD females, followed by NTg females and finally 3xTg-AD males, whose performance is low and does not improve with training. This difference persisted in the re-test at 16 months. In males, differences were also detected in the physical endurance and each training days, with significance in the age of the NTg animals and the effect of Re-test in 3xTg-AD and the NTg (see Figure 6A and Tables S1-S3). In addition, on the first day of training, it was observed that 3xTg-AD males showed differences in R and aging effect among naïve mice (see Figure 6B, and Tables S1-S3). On the second day of training, the difference in G at 12 months and the effect of aging in the naïve 3xTg-AD group stand out. The changes observed on the third day of the test were recorded at 12 months, where the G has statistical significance and the R only in NTg group. For females, physical endurance was higher in the 3xTg-AD group. The re-test 16 months group had high latencies (see Figure 6A and Table S2). Differences were observed on Day 1 and Day 3, with differences was in the 16 months re-test NTg group and aging effect in the 3xTg-AD group (see Figure 6B and Table S3).
On the other hand, differences in genotype and sex were detected in the 12 months group (see Figure 6A and Table S1). In the 3 days of training, differences in effect were detected, with G distinction only on the second day ( Figure 6B, and Table S1). Additionally, at 16 months in the re-test group, differences in G×S were recorded in physical endurance (see Figure 6A and Table S2). Days 2 and 3 showed differences in G×S, with no significant differences on the first day (see Figure 6B and Table S2). The re-test of this group corroborated the differences in G×S of the batch at 16 months (see Figure 6B and Table S2). However, at 16 months, groups of naïve mice did not show significant differences in this test. Yet, when comparing the re-test and naïve mice at 16 months, significant differences were detected between the group of males in physical endurance and the performance of Days 1 and 3 (see Figure 6A,B and Table S3). In addition, among the group of transgenic mice, differences in sex and re-test were detected between the groups, with the performance of the females being higher (see Figure 6A,B and Table S3). ANOVA, MRA-ANOVA, G, genotype effect, G** p < 0.01, G* p < 0.05. S, Sex effect, S*** p < 0.001***, S** p < 0.01**, S* p < 0.05. R, re-test effect, naïve 12 months vs. re-test 16 months, R*** p < 0.001***, R** p < 0.01**, R* p < 0.05*. G×S, genotype and sex effects, G×S** p < 0.01**, G×S* p < 0.05*. S×R, sex and re-test effects, S×R* p < 0.05*. Bonferroni post hoc test: g, genotype; s, sex; r: re-test, naïve 12 months vs. 16 months; n, naïve, naïve 16 months vs. re-test 16 months; $ expressed genotype, and # expressed sex differences between genotypes. The symbol ⦸ indicates the absence of the group, and m, month. ANOVA, MRA-ANOVA, G, genotype effect, G** p < 0.01, G* p < 0.05. S, Sex effect, S*** p < 0.001***, S** p < 0.01**, S* p < 0.05. R, re-test effect, naïve 12 months vs. re-test 16 months, R*** p < 0.001***, R** p < 0.01**, R* p < 0.05*. G×S, genotype and sex effects, G×S** p < 0.01**, G×S* p < 0.05*. S×R, sex and re-test effects, S×R* p < 0.05*. Bonferroni post hoc test: g, genotype; s, sex; r: re-test, naïve 12 months vs. 16 months; n, naïve, naïve 16 months vs. re-test 16 months; $ expressed genotype, and # expressed sex differences between genotypes. The symbol  Furthermore, considering MRA between the groups, we can differentiate the effect of G, S, and R in the day-by-day and trial-by-trial tests, Table S4 shows the statistical differences from Figure 6C. The MRA analysis between males on Day 1 showed differences in G and S, see Figure 6C. It was observed that the NTg retest males improve with the repetition of the trials as well as the 3xTg-AD, but these do so to a lesser extent, and both the 12 months and 16 months naïve males have lower performance than retest. On Day 2, the genotype effect was observed here. The naïve 12 months NTg mice and the retest 16 months show a high latency in the test that increases with the execution of the trials. Naïve 16 months 3xTg-AD mice show the best performance within this group. In addition, the MRA trial-by-trial showed the differences in each trial and the animals' G and R differences. Here, it is highlighted that the first day plays an important role in the retest and then the differences of genotype. Also, among the females, significant differences were recorded in MRA trial by trial, with the 3xTg-AD retest of 16 months being the ones with the highest performance during all the test days. On the first day of training, differences in performance were obtained between the naïve 12 m NTg females and their retest 16 months, with a higher latency between the 16 months 3xTg-AD retest ( Figure 6C). The second day of training did not record differences between the females, but on the third day, the highest performance of the 3xTg-AD retest 16 months was observed again.
Additionally, it is possible to differentiate females from males in the 3xTg-AD group, with females showing better performance in all tests. Thus, on Day 1 the mice differ in S and R. On the second day, the differences obtained on Day 1 are maintained, but the difference in the gender factor increases between the groups. On the third day, it is only possible to differentiate the gender factor between the groups. Specifically, the differences between the different factors have been identified in each trial. Thus, we can highlight specific differences be-tween the groups as detailed below on supplementary data for males and females. In addition, differences between 3xTg-AD males and females were detected in the following trials (see Figure 6C and Table S4).
Furthermore, corticosterone levels were correlated with different variables, detecting a different correlation between males and females. In males, a negative correlation with the muscle weight of the quadriceps and triceps surae stands out, and a positive correlation with the variables, phenotype scoring system, frailty score, cadence and physical endurance on the first day (quadriceps, r 2 = (-) 0.141, p = 0.008; triceps surae, r 2 = (-) 0.098, p = 0.03).   Phenotype scoring system, r 2 = 0.182, p = 0.002; frailty score, r 2 = 0.119, p = 0.016; Cadence, r 2 = 0.092, p = 0.036; Physical endurance day 1, r 2 = 0.190, p = 0.002), see Figure 8A-F. In females, a positive correlation between corticosterone with performance in the rotarod on total, the second and third day were detected (physical endurance-total, r 2 = 0.143, p = 0.039; physical endurance Day 2, r 2 = 0.157, p = 0.03, physical endurance Day 3, r 2 = 0.168, p = 0.024), see Figure 8G-I.  In different way, only in male, functional correlations with sarcopenia index were detected. Thus, sarcopenia index-quadriceps correlations with physical endurance Day 1 and Day 2 (sarcopenia index-quadriceps-physical endurance Day 1, r 2 = 0.190, p = 0.002. sarcopenia index-quadriceps-physical endurance Day 2, r 2 = 0.084, p = 0.048). In ad-dition, sarcopenia index-triceps surae correlation with the number of horizontal explorations (visited corners) (sarcopenia index-triceps surae-corners, r 2 = (−)0.099, p = 0.029), see Figure 9A-C.  There is the summary of results in Table 2.  On the other hand, males and females had a negative correlation between phenotype score system and functional variables. In the case of males, a negative correlation was detected between stride length and the phenotype scoring system (stride length-phenotype scoring system, r 2 = (−) 0.178, p = 0.003). In females, a negative correlation is observed with physical endurance-total (phenotype scoring system, r 2 = (−) 0.208, p = 0.011), see Figure 10A,B.  There is the summary of results in Table 2.  There is the summary of results in Table 2.  Triceps surae and sarcopenia index naïve 3xTg-AD females re-test females at 16 m High mortality, mostly among NTg female mice, rescued in longitudinal designs 3xTg-AD males at 12 m, and males, negative correlations between corticosterone and quadriceps, triceps surae; and positive correlations between corticosterone and phenotype score system, frailty score, cadence, and physical endurance Day 1. ales, positives correlated between corticosterone and physical endurance-total, physical endurance Days 2 and 3. ive correlations in males were detected between sarcopenia index-quadriceps and physical endurance on Days 1 and 2. In females, negative correlations were detected between sarcopenia index-triceps and horizontal activity. ative correlations in males were identified between phenotype score system and stride length, and in females' phenotype score system and physical endurance-total. According to the factors, genotype (G), sex (S), re-test (R) and naïve (N), a summary of the main results of this study is presented. It also includes the correlation's interactions. The symbol indicates increase, indicates decreases, and m, month.

Discussion
Recently, we developed a battery of psychomotor tests that include gait, neophobia and exploration, muscle strength, motor learning, physical resistance, and frailty status [33]. The results, in males, indicated that 3xTg-AD mice exhibit a more significant functional impairment in the quantitative variables of gait and exploratory activity than agematched NTg counterparts with normal aging. The presence of movement limitations and muscle weakness was determinant for the functional decline related to the stages of severity of the disease that worsened with age. In addition, we detected the presence of signs of physical frailty, which accompany the functional deterioration of these animals. The signs of sarcopenia were present in an advanced stage of AD [31,32]. Therefore, the present study was designed to investigate, for the first time, several aspects: (1) from a gender-medicine perspective, the impact of this functional impairment in 3xTg-AD females as compared to males; (2) the long-term effects of repeated test, either in longitudinal (the same set of animals at 12 and 16 months of age) or transversal (two different sets, pretested or naïve, at 16 months of age) designs, both in pathological and normal aging scenarios; 3) to include a phenotype of frailty and physical deterioration that may find a functional correlation with the biological status (HPA axis and sarcopenia), with nuances in male and female animals.

Survival
The survival curves on the cohorts of 191 animals allowed us to record higher mortality in females, being the group of NTg females the one that presented the highest number of deaths between 8-12 months of age. Interestingly, only females under the longitudinal design survived and achieved 16 months of age, while the group of naïve NTg females perished before reaching that old age, suggesting that repeated testing might have some protective effects. These results agree with our previous reports in these colonies, where high mortality rates associated with increased frailty were reported in females, and NTg exhibited increased mortality from 12 months of age [42]. In the case of 3xTg-AD mice, females that reached old age were survivors who overcame the disease's advanced neuropathological stages and exhibited lower behavioural differences with their NTg counterparts except for cognitive AD-hallmarks [47]. We have also described that, in male 3xTg-AD mice, an increase of mortality rates is associated with impairment in the neuroimmune-endocrine system compared to their females counterparts or the NTg genotype [48][49][50]. Noteworthy, we have recently reported survival bias and crosstalk between chronological and behavioral age in an APPswe model, where age-and genotype-sensitivity tests defined behavioral signatures in middle-aged, old, and long-lived mice with normal and AD-associated aging [51]. Therefore, the present work provides further evidence on sex and genotype-dependent differences in life expectancy and supports the key role of frailty and compensatory mechanisms as previously reported by our and other laboratories using different models of AD [29,[49][50][51][52].
in the re-test at 16 m PEER REVIEW 21 of 31 les, negative correlations between corticosterone and quadriceps, triceps surae; and positive correlations een corticosterone and phenotype score system, frailty score, cadence, and physical endurance Day 1. , positives correlated between corticosterone and physical endurance-total, physical endurance Days 2 and 3. correlations in males were detected between sarcopenia index-quadriceps and physical endurance on Days 1 and 2. emales, negative correlations were detected between sarcopenia index-triceps and horizontal activity. e correlations in males were identified between phenotype score system and stride length, and in females' phenotype score system and physical endurance-total. According to the factors, genotype (G), sex (S), re-test (R) and naïve (N), a summary of the main results of this study is presented. It also includes the correlation's interactions. The symbol indicates increase, indicates decreases, and m, month.

Discussion
Recently, we developed a battery of psychomotor tests that include gait, neophobia and exploration, muscle strength, motor learning, physical resistance, and frailty status [33]. The results, in males, indicated that 3xTg-AD mice exhibit a more significant functional impairment in the quantitative variables of gait and exploratory activity than agematched NTg counterparts with normal aging. The presence of movement limitations and muscle weakness was determinant for the functional decline related to the stages of severity of the disease that worsened with age. In addition, we detected the presence of signs of physical frailty, which accompany the functional deterioration of these animals. The signs of sarcopenia were present in an advanced stage of AD [31,32]. Therefore, the present study was designed to investigate, for the first time, several aspects: (1) from a gender-medicine perspective, the impact of this functional impairment in 3xTg-AD females as compared to males; (2) the long-term effects of repeated test, either in longitudinal (the same set of animals at 12 and 16 months of age) or transversal (two different sets, pretested or naïve, at 16 months of age) designs, both in pathological and normal aging scenarios; 3) to include a phenotype of frailty and physical deterioration that may find a functional correlation with the biological status (HPA axis and sarcopenia), with nuances in male and female animals.

Survival
The survival curves on the cohorts of 191 animals allowed us to record higher mortality in females, being the group of NTg females the one that presented the highest number of deaths between 8-12 months of age. Interestingly, only females under the longitudinal design survived and achieved 16 months of age, while the group of naïve NTg females perished before reaching that old age, suggesting that repeated testing might have some protective effects. These results agree with our previous reports in these colonies, where high mortality rates associated with increased frailty were reported in females, and NTg exhibited increased mortality from 12 months of age [42]. In the case of 3xTg-AD mice, females that reached old age were survivors who overcame the disease's advanced neuropathological stages and exhibited lower behavioural differences with their NTg counterparts except for cognitive AD-hallmarks [47]. We have also described that, in male 3xTg-AD mice, an increase of mortality rates is associated with impairment in the neuroimmune-endocrine system compared to their females counterparts or the NTg genotype [48][49][50]. Noteworthy, we have recently reported survival bias and crosstalk between chronological and behavioral age in an APPswe model, where age-and genotype-sensitivity tests defined behavioral signatures in middle-aged, old, and long-lived mice with normal and AD-associated aging [51]. Therefore, the present work provides further evidence on sex and genotype-dependent differences in life expectancy and supports the key role of frailty and compensatory mechanisms as previously reported by our and other laboratories using different models of AD [29,[49][50][51][52]. ales, negative correlations between corticosterone and quadriceps, triceps surae; and positive correlations ween corticosterone and phenotype score system, frailty score, cadence, and physical endurance Day 1. s, positives correlated between corticosterone and physical endurance-total, physical endurance Days 2 and 3. correlations in males were detected between sarcopenia index-quadriceps and physical endurance on Days 1 and 2. females, negative correlations were detected between sarcopenia index-triceps and horizontal activity. ve correlations in males were identified between phenotype score system and stride length, and in females' phenotype score system and physical endurance-total. According to the factors, genotype (G), sex (S), re-test (R) and naïve (N), a summary of the main results of this study is presented. It also includes the correlation's interactions. The symbol indicates increase, indicates decreases, and m, month.

Discussion
Recently, we developed a battery of psychomotor tests that include gait, neophobia and exploration, muscle strength, motor learning, physical resistance, and frailty status [33]. The results, in males, indicated that 3xTg-AD mice exhibit a more significant functional impairment in the quantitative variables of gait and exploratory activity than agematched NTg counterparts with normal aging. The presence of movement limitations and muscle weakness was determinant for the functional decline related to the stages of severity of the disease that worsened with age. In addition, we detected the presence of signs of physical frailty, which accompany the functional deterioration of these animals. The signs of sarcopenia were present in an advanced stage of AD [31,32]. Therefore, the present study was designed to investigate, for the first time, several aspects: (1) from a gender-medicine perspective, the impact of this functional impairment in 3xTg-AD females as compared to males; (2) the long-term effects of repeated test, either in longitudinal (the same set of animals at 12 and 16 months of age) or transversal (two different sets, pretested or naïve, at 16 months of age) designs, both in pathological and normal aging scenarios; 3) to include a phenotype of frailty and physical deterioration that may find a functional correlation with the biological status (HPA axis and sarcopenia), with nuances in male and female animals. The survival curves on the cohorts of 191 animals allowed us to record higher mortality in females, being the group of NTg females the one that presented the highest number of deaths between 8-12 months of age. Interestingly, only females under the longitudinal design survived and achieved 16 months of age, while the group of naïve NTg females perished before reaching that old age, suggesting that repeated testing might have some protective effects. These results agree with our previous reports in these colonies, where high mortality rates associated with increased frailty were reported in females, and NTg exhibited increased mortality from 12 months of age [42]. In the case of 3xTg-AD mice, females that reached old age were survivors who overcame the disease's advanced neuropathological stages and exhibited lower behavioural differences with their NTg counterparts except for cognitive AD-hallmarks [47]. We have also described that, in male 3xTg-AD mice, an increase of mortality rates is associated with impairment in the neuroimmune-endocrine system compared to their females counterparts or the NTg genotype [48][49][50]. Noteworthy, we have recently reported survival bias and crosstalk between chronological and behavioral age in an APPswe model, where age-and genotype-sensitivity tests defined behavioral signatures in middle-aged, old, and long-lived mice with normal and AD-associated aging [51]. Therefore, the present work provides further evidence on sex and genotype-dependent differences in life expectancy and supports the key role of frailty and compensatory mechanisms as previously reported by our and other laboratories using different models of AD [29,[49][50][51][52]. ales, negative correlations between corticosterone and quadriceps, triceps surae; and positive correlations tween corticosterone and phenotype score system, frailty score, cadence, and physical endurance Day 1. s, positives correlated between corticosterone and physical endurance-total, physical endurance Days 2 and 3. e correlations in males were detected between sarcopenia index-quadriceps and physical endurance on Days 1 and 2. females, negative correlations were detected between sarcopenia index-triceps and horizontal activity. ive correlations in males were identified between phenotype score system and stride length, and in females' phenotype score system and physical endurance-total. According to the factors, genotype (G), sex (S), re-test (R) and naïve (N), a summary of the main results of this study is presented. It also includes the correlation's interactions. The symbol indicates increase, indicates decreases, and m, month.

Discussion
Recently, we developed a battery of psychomotor tests that include gait, neophobia and exploration, muscle strength, motor learning, physical resistance, and frailty status [33]. The results, in males, indicated that 3xTg-AD mice exhibit a more significant functional impairment in the quantitative variables of gait and exploratory activity than agematched NTg counterparts with normal aging. The presence of movement limitations and muscle weakness was determinant for the functional decline related to the stages of severity of the disease that worsened with age. In addition, we detected the presence of signs of physical frailty, which accompany the functional deterioration of these animals. The signs of sarcopenia were present in an advanced stage of AD [31,32]. Therefore, the present study was designed to investigate, for the first time, several aspects: (1) from a gender-medicine perspective, the impact of this functional impairment in 3xTg-AD females as compared to males; (2) the long-term effects of repeated test, either in longitudinal (the same set of animals at 12 and 16 months of age) or transversal (two different sets, pretested or naïve, at 16 months of age) designs, both in pathological and normal aging scenarios; 3) to include a phenotype of frailty and physical deterioration that may find a functional correlation with the biological status (HPA axis and sarcopenia), with nuances in male and female animals. The survival curves on the cohorts of 191 animals allowed us to record higher mortality in females, being the group of NTg females the one that presented the highest number of deaths between 8-12 months of age. Interestingly, only females under the longitudinal design survived and achieved 16 months of age, while the group of naïve NTg females perished before reaching that old age, suggesting that repeated testing might have some protective effects. These results agree with our previous reports in these colonies, where high mortality rates associated with increased frailty were reported in females, and NTg exhibited increased mortality from 12 months of age [42]. In the case of 3xTg-AD mice, females that reached old age were survivors who overcame the disease's advanced neuropathological stages and exhibited lower behavioural differences with their NTg counterparts except for cognitive AD-hallmarks [47]. We have also described that, in male 3xTg-AD mice, an increase of mortality rates is associated with impairment in the neuroimmune-endocrine system compared to their females counterparts or the NTg genotype [48][49][50]. Noteworthy, we have recently reported survival bias and crosstalk between chronological and behavioral age in an APPswe model, where age-and genotype-sensitivity tests defined behavioral signatures in middle-aged, old, and long-lived mice with normal and AD-associated aging [51]. Therefore, the present work provides further evidence on sex and genotype-dependent differences in life expectancy and supports the key role of frailty and compensatory mechanisms as previously reported by our and other laboratories using different models of AD [29,[49][50][51][52]. les, negative correlations between corticosterone and quadriceps, triceps surae; and positive correlations een corticosterone and phenotype score system, frailty score, cadence, and physical endurance Day 1. , positives correlated between corticosterone and physical endurance-total, physical endurance Days 2 and 3. correlations in males were detected between sarcopenia index-quadriceps and physical endurance on Days 1 and 2. emales, negative correlations were detected between sarcopenia index-triceps and horizontal activity. e correlations in males were identified between phenotype score system and stride length, and in females' phenotype score system and physical endurance-total. According to the factors, genotype (G), sex (S), re-test (R) and naïve (N), a summary of the main results of this study is presented. It also includes the correlation's interactions. The symbol indicates increase, indicates decreases, and m, month.

Discussion
Recently, we developed a battery of psychomotor tests that include gait, neophobia and exploration, muscle strength, motor learning, physical resistance, and frailty status [33]. The results, in males, indicated that 3xTg-AD mice exhibit a more significant functional impairment in the quantitative variables of gait and exploratory activity than agematched NTg counterparts with normal aging. The presence of movement limitations and muscle weakness was determinant for the functional decline related to the stages of severity of the disease that worsened with age. In addition, we detected the presence of signs of physical frailty, which accompany the functional deterioration of these animals. The signs of sarcopenia were present in an advanced stage of AD [31,32]. Therefore, the present study was designed to investigate, for the first time, several aspects: (1) from a gender-medicine perspective, the impact of this functional impairment in 3xTg-AD females as compared to males; (2) the long-term effects of repeated test, either in longitudinal (the same set of animals at 12 and 16 months of age) or transversal (two different sets, pretested or naïve, at 16 months of age) designs, both in pathological and normal aging scenarios; 3) to include a phenotype of frailty and physical deterioration that may find a functional correlation with the biological status (HPA axis and sarcopenia), with nuances in male and female animals.

Survival
The survival curves on the cohorts of 191 animals allowed us to record higher mortality in females, being the group of NTg females the one that presented the highest number of deaths between 8-12 months of age. Interestingly, only females under the longitudinal design survived and achieved 16 months of age, while the group of naïve NTg females perished before reaching that old age, suggesting that repeated testing might have some protective effects. These results agree with our previous reports in these colonies, where high mortality rates associated with increased frailty were reported in females, and NTg exhibited increased mortality from 12 months of age [42]. In the case of 3xTg-AD mice, females that reached old age were survivors who overcame the disease's advanced neuropathological stages and exhibited lower behavioural differences with their NTg counterparts except for cognitive AD-hallmarks [47]. We have also described that, in male 3xTg-AD mice, an increase of mortality rates is associated with impairment in the neuroimmune-endocrine system compared to their females counterparts or the NTg genotype [48][49][50]. Noteworthy, we have recently reported survival bias and crosstalk between chronological and behavioral age in an APPswe model, where age-and genotype-sensitivity tests defined behavioral signatures in middle-aged, old, and long-lived mice with normal and AD-associated aging [51]. Therefore, the present work provides further evidence on sex and genotype-dependent differences in life expectancy and supports the key role of frailty and compensatory mechanisms as previously reported by our and other laboratories using different models of AD [29,[49][50][51][52]. Naïve

Correlation's interactions
In males, negative correlations between corticosterone and quadriceps, triceps surae; and positive correlations between corticosterone and phenotype score system, frailty score, cadence, and physical endurance Day 1. Females, positives correlated between corticosterone and physical endurance-total, physical endurance Days 2 and 3. Positive correlations in males were detected between sarcopenia index-quadriceps and physical endurance on Days 1 and 2. In females, negative correlations were detected between sarcopenia index-triceps and horizontal activity. Negative correlations in males were identified between phenotype score system and stride length, and in females' phenotype score system and physical endurance-total. According to the factors, genotype (G), sex (S), re-test (R) and naïve (N), a summary of the main results of this study is presented. It also includes the correlation's interactions. The symbol indicates increase, indicates decreases, and m, month.

Discussion
Recently, we developed a battery of psychomotor tests that include gait, neophobia and exploration, muscle strength, motor learning, physical resistance, and frailty status [33]. The results, in males, indicated that 3xTg-AD mice exhibit a more significant functional impairment in the quantitative variables of gait and exploratory activity than agematched NTg counterparts with normal aging. The presence of movement limitations and muscle weakness was determinant for the functional decline related to the stages of severity of the disease that worsened with age. In addition, we detected the presence of signs of physical frailty, which accompany the functional deterioration of these animals. The signs of sarcopenia were present in an advanced stage of AD [31,32]. Therefore, the present study was designed to investigate, for the first time, several aspects: (1) from a gender-medicine perspective, the impact of this functional impairment in 3xTg-AD females as compared to males; (2) the long-term effects of repeated test, either in longitudinal (the same set of animals at 12 and 16 months of age) or transversal (two different sets, pretested or naïve, at 16 months of age) designs, both in pathological and normal aging scenarios; 3) to include a phenotype of frailty and physical deterioration that may find a functional correlation with the biological status (HPA axis and sarcopenia), with nuances in male and female animals. The survival curves on the cohorts of 191 animals allowed us to record higher mortality in females, being the group of NTg females the one that presented the highest number of deaths between 8-12 months of age. Interestingly, only females under the longitudinal design survived and achieved 16 months of age, while the group of naïve NTg females perished before reaching that old age, suggesting that repeated testing might have some protective effects. These results agree with our previous reports in these colonies, where high mortality rates associated with increased frailty were reported in females, and NTg exhibited increased mortality from 12 months of age [42]. In the case of 3xTg-AD mice, females that reached old age were survivors who overcame the disease's advanced neuropathological stages and exhibited lower behavioural differences with their NTg counterparts except for cognitive AD-hallmarks [47]. We have also described that, in male 3xTg-AD mice, an increase of mortality rates is associated with impairment in the neuroimmune-endocrine system compared to their females counterparts or the NTg genotype [48][49][50]. Noteworthy, we have recently reported survival bias and crosstalk between chronological and behavioral age in an APPswe model, where age-and genotype-sensitivity tests defined behavioral signatures in middle-aged, old, and long-lived mice with normal and AD-associated aging [51]. Therefore, the present work provides further evidence on sex and genotype-dependent differences in life expectancy and supports the key role of frailty and compensatory mechanisms as previously reported by our and other laboratories using different models of AD [29,[49][50][51][52].

Correlation's interactions
In males, negative correlations between corticosterone and quadriceps, triceps surae; and positive between corticosterone and phenotype score system, frailty score, cadence, and physical enduran Females, positives correlated between corticosterone and physical endurance-total, physical enduran 3. Positive correlations in males were detected between sarcopenia index-quadriceps and physical endu 1 and 2. In females, negative correlations were detected between sarcopenia index-triceps and horizonta Negative correlations in males were identified between phenotype score system and stride length, an phenotype score system and physical endurance-total. According to the factors, genotype (G), sex (S), re-test (R) and naïve (N), a summary results of this study is presented. It also includes the correlation's interactions. The sy indicates increase, indicates decreases, and m, month.

Discussion
Recently, we developed a battery of psychomotor tests that include gai and exploration, muscle strength, motor learning, physical resistance, and [33]. The results, in males, indicated that 3xTg-AD mice exhibit a more sign tional impairment in the quantitative variables of gait and exploratory activ matched NTg counterparts with normal aging. The presence of movement lim muscle weakness was determinant for the functional decline related to the verity of the disease that worsened with age. In addition, we detected the pres of physical frailty, which accompany the functional deterioration of these signs of sarcopenia were present in an advanced stage of AD [31,32]. There sent study was designed to investigate, for the first time, several aspects: (1 der-medicine perspective, the impact of this functional impairment in 3xTg as compared to males; (2) the long-term effects of repeated test, either in long same set of animals at 12 and 16 months of age) or transversal (two differ tested or naïve, at 16 months of age) designs, both in pathological and norm narios; 3) to include a phenotype of frailty and physical deterioration that ma tional correlation with the biological status (HPA axis and sarcopenia), wit male and female animals. The survival curves on the cohorts of 191 animals allowed us to record tality in females, being the group of NTg females the one that presented the h ber of deaths between 8-12 months of age. Interestingly, only females under dinal design survived and achieved 16 months of age, while the group of n males perished before reaching that old age, suggesting that repeated testing some protective effects. These results agree with our previous reports in th where high mortality rates associated with increased frailty were reported in NTg exhibited increased mortality from 12 months of age [42]. In the case mice, females that reached old age were survivors who overcame the diseas neuropathological stages and exhibited lower behavioural differences wit counterparts except for cognitive AD-hallmarks [47]. We have also described 3xTg-AD mice, an increase of mortality rates is associated with impairment immune-endocrine system compared to their females counterparts or the N [48][49][50]. Noteworthy, we have recently reported survival bias and crosst chronological and behavioral age in an APPswe model, where age-and gen tivity tests defined behavioral signatures in middle-aged, old, and long-live normal and AD-associated aging [51]. Therefore, the present work provides dence on sex and genotype-dependent differences in life expectancy and sup role of frailty and compensatory mechanisms as previously reported by o laboratories using different models of AD [29,[49][50][51][52].

Correlation's interactions
In males, negative correlations between corticosterone and quadriceps, triceps surae; and positive between corticosterone and phenotype score system, frailty score, cadence, and physical endura Females, positives correlated between corticosterone and physical endurance-total, physical endura 3. Positive correlations in males were detected between sarcopenia index-quadriceps and physical end 1 and 2. In females, negative correlations were detected between sarcopenia index-triceps and horizont Negative correlations in males were identified between phenotype score system and stride length, a phenotype score system and physical endurance-total. According to the factors, genotype (G), sex (S), re-test (R) and naïve (N), a summary results of this study is presented. It also includes the correlation's interactions. The indicates increase, indicates decreases, and m, month.

Discussion
Recently, we developed a battery of psychomotor tests that include ga and exploration, muscle strength, motor learning, physical resistance, and [33]. The results, in males, indicated that 3xTg-AD mice exhibit a more sig tional impairment in the quantitative variables of gait and exploratory acti matched NTg counterparts with normal aging. The presence of movement li muscle weakness was determinant for the functional decline related to th verity of the disease that worsened with age. In addition, we detected the pre of physical frailty, which accompany the functional deterioration of these signs of sarcopenia were present in an advanced stage of AD [31,32]. Ther sent study was designed to investigate, for the first time, several aspects: (1 der-medicine perspective, the impact of this functional impairment in 3xT as compared to males; (2) the long-term effects of repeated test, either in lon same set of animals at 12 and 16 months of age) or transversal (two diffe tested or naïve, at 16 months of age) designs, both in pathological and norm narios; 3) to include a phenotype of frailty and physical deterioration that ma tional correlation with the biological status (HPA axis and sarcopenia), wi male and female animals. The survival curves on the cohorts of 191 animals allowed us to recor tality in females, being the group of NTg females the one that presented the ber of deaths between 8-12 months of age. Interestingly, only females unde dinal design survived and achieved 16 months of age, while the group of males perished before reaching that old age, suggesting that repeated testin some protective effects. These results agree with our previous reports in t where high mortality rates associated with increased frailty were reported in NTg exhibited increased mortality from 12 months of age [42]. In the cas mice, females that reached old age were survivors who overcame the disea neuropathological stages and exhibited lower behavioural differences w counterparts except for cognitive AD-hallmarks [47]. We have also describe 3xTg-AD mice, an increase of mortality rates is associated with impairment immune-endocrine system compared to their females counterparts or the N [48][49][50]. Noteworthy, we have recently reported survival bias and cross chronological and behavioral age in an APPswe model, where age-and ge tivity tests defined behavioral signatures in middle-aged, old, and long-liv normal and AD-associated aging [51]. Therefore, the present work provide dence on sex and genotype-dependent differences in life expectancy and sup role of frailty and compensatory mechanisms as previously reported by o laboratories using different models of AD [29,[49][50][51][52].

Correlation's interactions
In males, negative correlations between corticosterone and quadriceps, triceps surae; and positive between corticosterone and phenotype score system, frailty score, cadence, and physical endura Females, positives correlated between corticosterone and physical endurance-total, physical endura 3. Positive correlations in males were detected between sarcopenia index-quadriceps and physical end 1 and 2. In females, negative correlations were detected between sarcopenia index-triceps and horizont Negative correlations in males were identified between phenotype score system and stride length, a phenotype score system and physical endurance-total. According to the factors, genotype (G), sex (S), re-test (R) and naïve (N), a summary results of this study is presented. It also includes the correlation's interactions. The indicates increase, indicates decreases, and m, month.

Discussion
Recently, we developed a battery of psychomotor tests that include ga and exploration, muscle strength, motor learning, physical resistance, and [33]. The results, in males, indicated that 3xTg-AD mice exhibit a more sig tional impairment in the quantitative variables of gait and exploratory acti matched NTg counterparts with normal aging. The presence of movement li muscle weakness was determinant for the functional decline related to th verity of the disease that worsened with age. In addition, we detected the pre of physical frailty, which accompany the functional deterioration of these signs of sarcopenia were present in an advanced stage of AD [31,32]. Ther sent study was designed to investigate, for the first time, several aspects: ( der-medicine perspective, the impact of this functional impairment in 3xT as compared to males; (2) the long-term effects of repeated test, either in lon same set of animals at 12 and 16 months of age) or transversal (two diffe tested or naïve, at 16 months of age) designs, both in pathological and norm narios; 3) to include a phenotype of frailty and physical deterioration that m tional correlation with the biological status (HPA axis and sarcopenia), w male and female animals. The survival curves on the cohorts of 191 animals allowed us to recor tality in females, being the group of NTg females the one that presented the ber of deaths between 8-12 months of age. Interestingly, only females und dinal design survived and achieved 16 months of age, while the group of males perished before reaching that old age, suggesting that repeated testin some protective effects. These results agree with our previous reports in t where high mortality rates associated with increased frailty were reported in NTg exhibited increased mortality from 12 months of age [42]. In the cas mice, females that reached old age were survivors who overcame the disea neuropathological stages and exhibited lower behavioural differences w counterparts except for cognitive AD-hallmarks [47]. We have also describe 3xTg-AD mice, an increase of mortality rates is associated with impairmen immune-endocrine system compared to their females counterparts or the N [48][49][50]. Noteworthy, we have recently reported survival bias and cros chronological and behavioral age in an APPswe model, where age-and ge tivity tests defined behavioral signatures in middle-aged, old, and long-liv normal and AD-associated aging [51]. Therefore, the present work provid dence on sex and genotype-dependent differences in life expectancy and su role of frailty and compensatory mechanisms as previously reported by laboratories using different models of AD [29,[49][50][51][52].

Correlation's interactions
In males, negative correlations between corticosterone and quadrice between corticosterone and phenotype score system, frailty score, Females, positives correlated between corticosterone and physical end 3. Positive correlations in males were detected between sarcopenia index 1 and 2. In females, negative correlations were detected between sarcopen Negative correlations in males were identified between phenotype sco phenotype score system and physical end According to the factors, genotype (G), sex (S), re-test results of this study is presented. It also includes the c indicates increase, indicates decreases, and m, mon

Discussion
Recently, we developed a battery of psycho and exploration, muscle strength, motor learnin [33]. The results, in males, indicated that 3xTg-A tional impairment in the quantitative variables o matched NTg counterparts with normal aging. Th muscle weakness was determinant for the funct verity of the disease that worsened with age. In ad of physical frailty, which accompany the functi signs of sarcopenia were present in an advanced sent study was designed to investigate, for the f der-medicine perspective, the impact of this fun as compared to males; (2) the long-term effects of same set of animals at 12 and 16 months of age tested or naïve, at 16 months of age) designs, bo narios; 3) to include a phenotype of frailty and ph tional correlation with the biological status (HP male and female animals.

Survival
The survival curves on the cohorts of 191 an tality in females, being the group of NTg females ber of deaths between 8-12 months of age. Intere dinal design survived and achieved 16 months o males perished before reaching that old age, sug some protective effects. These results agree with where high mortality rates associated with increa NTg exhibited increased mortality from 12 mon mice, females that reached old age were survivo neuropathological stages and exhibited lower counterparts except for cognitive AD-hallmarks [ 3xTg-AD mice, an increase of mortality rates is a immune-endocrine system compared to their fem [48][49][50]. Noteworthy, we have recently reporte chronological and behavioral age in an APPswe tivity tests defined behavioral signatures in mid normal and AD-associated aging [51]. Therefore dence on sex and genotype-dependent difference role of frailty and compensatory mechanisms a laboratories using different models of AD [

Correlation's interactions
In males, negative correlations between corticosterone and quadriceps, triceps surae; and positiv between corticosterone and phenotype score system, frailty score, cadence, and physical endur Females, positives correlated between corticosterone and physical endurance-total, physical endura 3. Positive correlations in males were detected between sarcopenia index-quadriceps and physical end 1 and 2. In females, negative correlations were detected between sarcopenia index-triceps and horizon Negative correlations in males were identified between phenotype score system and stride length, phenotype score system and physical endurance-total. According to the factors, genotype (G), sex (S), re-test (R) and naïve (N), a summar results of this study is presented. It also includes the correlation's interactions. The indicates increase, indicates decreases, and m, month.

Discussion
Recently, we developed a battery of psychomotor tests that include g and exploration, muscle strength, motor learning, physical resistance, and [33]. The results, in males, indicated that 3xTg-AD mice exhibit a more si tional impairment in the quantitative variables of gait and exploratory act matched NTg counterparts with normal aging. The presence of movement l muscle weakness was determinant for the functional decline related to th verity of the disease that worsened with age. In addition, we detected the pr of physical frailty, which accompany the functional deterioration of thes signs of sarcopenia were present in an advanced stage of AD [31,32]. Ther sent study was designed to investigate, for the first time, several aspects: ( der-medicine perspective, the impact of this functional impairment in 3xT as compared to males; (2) the long-term effects of repeated test, either in lon same set of animals at 12 and 16 months of age) or transversal (two diffe tested or naïve, at 16 months of age) designs, both in pathological and nor narios; 3) to include a phenotype of frailty and physical deterioration that m tional correlation with the biological status (HPA axis and sarcopenia), w male and female animals.

Survival
The survival curves on the cohorts of 191 animals allowed us to recor tality in females, being the group of NTg females the one that presented the ber of deaths between 8-12 months of age. Interestingly, only females und dinal design survived and achieved 16 months of age, while the group of males perished before reaching that old age, suggesting that repeated testi some protective effects. These results agree with our previous reports in

Correlation's interactions
In males, negative correlations between corticosterone and quadricep between corticosterone and phenotype score system, frailty score, c Females, positives correlated between corticosterone and physical endu 3. Positive correlations in males were detected between sarcopenia index-q 1 and 2. In females, negative correlations were detected between sarcopenia Negative correlations in males were identified between phenotype scor phenotype score system and physical endu According to the factors, genotype (G), sex (S), re-test (R results of this study is presented. It also includes the co indicates increase, indicates decreases, and m, mont

Discussion
Recently, we developed a battery of psychom and exploration, muscle strength, motor learning [33]. The results, in males, indicated that 3xTg-AD tional impairment in the quantitative variables of matched NTg counterparts with normal aging. The muscle weakness was determinant for the functio verity of the disease that worsened with age. In add of physical frailty, which accompany the function signs of sarcopenia were present in an advanced sent study was designed to investigate, for the fir der-medicine perspective, the impact of this func as compared to males; (2) the long-term effects of r same set of animals at 12 and 16 months of age) tested or naïve, at 16 months of age) designs, both narios; 3) to include a phenotype of frailty and phy tional correlation with the biological status (HPA male and female animals.

Survival
The survival curves on the cohorts of 191 ani tality in females, being the group of NTg females t ber of deaths between 8-12 months of age. Interes dinal design survived and achieved 16 months of males perished before reaching that old age, sugg some protective effects. These results agree with where high mortality rates associated with increas NTg exhibited increased mortality from 12 mont mice, females that reached old age were survivors ales, negative correlations between corticosterone and quadriceps, triceps surae; and positive correlations ween corticosterone and phenotype score system, frailty score, cadence, and physical endurance Day 1. s, positives correlated between corticosterone and physical endurance-total, physical endurance Days 2 and 3. correlations in males were detected between sarcopenia index-quadriceps and physical endurance on Days 1 and 2. females, negative correlations were detected between sarcopenia index-triceps and horizontal activity. ve correlations in males were identified between phenotype score system and stride length, and in females' phenotype score system and physical endurance-total. According to the factors, genotype (G), sex (S), re-test (R) and naïve (N), a summary of the main results of this study is presented. It also includes the correlation's interactions. The symbol indicates increase, indicates decreases, and m, month.

Discussion
Recently, we developed a battery of psychomotor tests that include gait, neophobia and exploration, muscle strength, motor learning, physical resistance, and frailty status [33]. The results, in males, indicated that 3xTg-AD mice exhibit a more significant functional impairment in the quantitative variables of gait and exploratory activity than agematched NTg counterparts with normal aging. The presence of movement limitations and muscle weakness was determinant for the functional decline related to the stages of severity of the disease that worsened with age. In addition, we detected the presence of signs of physical frailty, which accompany the functional deterioration of these animals. The signs of sarcopenia were present in an advanced stage of AD [31,32]. Therefore, the present study was designed to investigate, for the first time, several aspects: (1) from a gender-medicine perspective, the impact of this functional impairment in 3xTg-AD females as compared to males; (2) the long-term effects of repeated test, either in longitudinal (the same set of animals at 12 and 16 months of age) or transversal (two different sets, pretested or naïve, at 16 months of age) designs, both in pathological and normal aging scenarios; 3) to include a phenotype of frailty and physical deterioration that may find a functional correlation with the biological status (HPA axis and sarcopenia), with nuances in male and female animals. The survival curves on the cohorts of 191 animals allowed us to record higher mortality in females, being the group of NTg females the one that presented the highest number of deaths between 8-12 months of age. Interestingly, only females under the longitudinal design survived and achieved 16 months of age, while the group of naïve NTg fe-3xTg-AD males at 12 m and 16 m in the re-test group.
OR PEER REVIEW 21 of 31 males, negative correlations between corticosterone and quadriceps, triceps surae; and positive correlations etween corticosterone and phenotype score system, frailty score, cadence, and physical endurance Day 1. les, positives correlated between corticosterone and physical endurance-total, physical endurance Days 2 and 3. ve correlations in males were detected between sarcopenia index-quadriceps and physical endurance on Days 1 and 2. n females, negative correlations were detected between sarcopenia index-triceps and horizontal activity. tive correlations in males were identified between phenotype score system and stride length, and in females' phenotype score system and physical endurance-total. According to the factors, genotype (G), sex (S), re-test (R) and naïve (N), a summary of the main results of this study is presented. It also includes the correlation's interactions. The symbol indicates increase, indicates decreases, and m, month.

Discussion
Recently, we developed a battery of psychomotor tests that include gait, neophobia and exploration, muscle strength, motor learning, physical resistance, and frailty status [33]. The results, in males, indicated that 3xTg-AD mice exhibit a more significant functional impairment in the quantitative variables of gait and exploratory activity than agematched NTg counterparts with normal aging. The presence of movement limitations and muscle weakness was determinant for the functional decline related to the stages of severity of the disease that worsened with age. In addition, we detected the presence of signs of physical frailty, which accompany the functional deterioration of these animals. The signs of sarcopenia were present in an advanced stage of AD [31,32]. Therefore, the present study was designed to investigate, for the first time, several aspects: (1) from a gender-medicine perspective, the impact of this functional impairment in 3xTg-AD females as compared to males; (2) the long-term effects of repeated test, either in longitudinal (the same set of animals at 12 and 16 months of age) or transversal (two different sets, pretested or naïve, at 16 months of age) designs, both in pathological and normal aging scenarios; 3) to include a phenotype of frailty and physical deterioration that may find a functional correlation with the biological status (HPA axis and sarcopenia), with nuances in male and female animals. The survival curves on the cohorts of 191 animals allowed us to record higher mortality in females, being the group of NTg females the one that presented the highest num- les, negative correlations between corticosterone and quadriceps, triceps surae; and positive correlations een corticosterone and phenotype score system, frailty score, cadence, and physical endurance Day 1. , positives correlated between corticosterone and physical endurance-total, physical endurance Days 2 and 3. correlations in males were detected between sarcopenia index-quadriceps and physical endurance on Days 1 and 2. emales, negative correlations were detected between sarcopenia index-triceps and horizontal activity. e correlations in males were identified between phenotype score system and stride length, and in females' phenotype score system and physical endurance-total. According to the factors, genotype (G), sex (S), re-test (R) and naïve (N), a summary of the main results of this study is presented. It also includes the correlation's interactions. The symbol indicates increase, indicates decreases, and m, month.

Discussion
Recently, we developed a battery of psychomotor tests that include gait, neophobia and exploration, muscle strength, motor learning, physical resistance, and frailty status [33]. The results, in males, indicated that 3xTg-AD mice exhibit a more significant functional impairment in the quantitative variables of gait and exploratory activity than agematched NTg counterparts with normal aging. The presence of movement limitations and muscle weakness was determinant for the functional decline related to the stages of severity of the disease that worsened with age. In addition, we detected the presence of signs of physical frailty, which accompany the functional deterioration of these animals. The signs of sarcopenia were present in an advanced stage of AD [31,32]. Therefore, the present study was designed to investigate, for the first time, several aspects: (1) from a gender-medicine perspective, the impact of this functional impairment in 3xTg-AD females as compared to males; (2) the long-term effects of repeated test, either in longitudinal (the same set of animals at 12 and 16 months of age) or transversal (two different sets, pretested or naïve, at 16 months of age) designs, both in pathological and normal aging scenarios; 3) to include a phenotype of frailty and physical deterioration that may find a functional correlation with the biological status (HPA axis and sarcopenia), with nuances in male and female animals.

Correlation's interactions
In males, negative correlations between corticosterone and quadricep between corticosterone and phenotype score system, frailty score, c Females, positives correlated between corticosterone and physical endu 3. Positive correlations in males were detected between sarcopenia index-q 1 and 2. In females, negative correlations were detected between sarcopeni Negative correlations in males were identified between phenotype scor phenotype score system and physical end According to the factors, genotype (G), sex (S), re-test ( results of this study is presented. It also includes the co indicates increase, indicates decreases, and m, mon

Discussion
Recently, we developed a battery of psychom and exploration, muscle strength, motor learning [33]. The results, in males, indicated that 3xTg-A tional impairment in the quantitative variables of matched NTg counterparts with normal aging. Th muscle weakness was determinant for the functi verity of the disease that worsened with age. In ad of physical frailty, which accompany the functio signs of sarcopenia were present in an advanced sent study was designed to investigate, for the fir der-medicine perspective, the impact of this func as compared to males; (2) the long-term effects of same set of animals at 12 and 16 months of age) tested or naïve, at 16 months of age) designs, bot narios; 3) to include a phenotype of frailty and phy tional correlation with the biological status (HPA male and female animals.

Correlation's interactions
In males, negative correlations between corticosterone and quadriceps, triceps surae; and positive correlations between corticosterone and phenotype score system, frailty score, cadence, and physical endurance Day 1. Females, positives correlated between corticosterone and physical endurance-total, physical endurance Days 2 and 3. Positive correlations in males were detected between sarcopenia index-quadriceps and physical endurance on Days 1 and 2. In females, negative correlations were detected between sarcopenia index-triceps and horizontal activity. Negative correlations in males were identified between phenotype score system and stride length, and in females' phenotype score system and physical endurance-total. According to the factors, genotype (G), sex (S), re-test (R) and naïve (N), a summary of the main results of this study is presented. It also includes the correlation's interactions. The symbol indicates increase, indicates decreases, and m, month.

Discussion
Recently, we developed a battery of psychomotor tests that include gait, neophobia and exploration, muscle strength, motor learning, physical resistance, and frailty status [33]. The results, in males, indicated that 3xTg-AD mice exhibit a more significant functional impairment in the quantitative variables of gait and exploratory activity than agematched NTg counterparts with normal aging. The presence of movement limitations and muscle weakness was determinant for the functional decline related to the stages of severity of the disease that worsened with age. In addition, we detected the presence of signs of physical frailty, which accompany the functional deterioration of these animals. The signs of sarcopenia were present in an advanced stage of AD [31,32]. Therefore, the present study was designed to investigate, for the first time, several aspects: (1) from a gender-medicine perspective, the impact of this functional impairment in 3xTg-AD females as compared to males; (2) the long-term effects of repeated test, either in longitudinal (the same set of animals at 12 and 16 months of age) or transversal (two different sets, pretested or naïve, at 16 months of age) designs, both in pathological and normal aging scenarios; 3) to include a phenotype of frailty and physical deterioration that may find a func-

Correlation's interactions
In males, negative correlations between corticosterone and quadricep between corticosterone and phenotype score system, frailty score, c Females, positives correlated between corticosterone and physical endu 3. Positive correlations in males were detected between sarcopenia index-q 1 and 2. In females, negative correlations were detected between sarcopenia Negative correlations in males were identified between phenotype scor phenotype score system and physical endu According to the factors, genotype (G), sex (S), re-test (R results of this study is presented. It also includes the co indicates increase, indicates decreases, and m, mont

Discussion
Recently, we developed a battery of psychom and exploration, muscle strength, motor learning [33]. The results, in males, indicated that 3xTg-AD tional impairment in the quantitative variables of matched NTg counterparts with normal aging. The muscle weakness was determinant for the functio verity of the disease that worsened with age. In add of physical frailty, which accompany the functio signs of sarcopenia were present in an advanced sent study was designed to investigate, for the fir der-medicine perspective, the impact of this func as compared to males; (2) the long-term effects of r same set of animals at 12 and 16 months of age) tested or naïve, at 16 months of age) designs, both Triceps surae and sarcopenia index naïve 3xTg-AD females re-test females at 16 m Survival High mortality, mostly among NTg female mice, rescued in longitudinal designs

Correlation's interactions
In males, negative correlations between corticosterone and quadriceps, triceps surae; and positive correlations between corticosterone and phenotype score system, frailty score, cadence, and physical endurance Day 1. Females, positives correlated between corticosterone and physical endurance-total, physical endurance Days 2 and 3. Positive correlations in males were detected between sarcopenia index-quadriceps and physical endurance on Days 1 and 2.
In females, negative correlations were detected between sarcopenia index-triceps and horizontal activity. Negative correlations in males were identified between phenotype score system and stride length, and in females' phenotype score system and physical endurance-total.
According to the factors, genotype (G), sex (S), re-test (R) and naïve (N), a summary of the main results of this study is presented. It also includes the correlation's interactions.

Correlation's interactions
In males, negative correlations between corticosteron between corticosterone and phenotype score system Females, positives correlated between corticosterone an Positive correlations in males were detected between sa 1 In females, negative correlations were detected bet Negative correlations in males were identified between phenotype score system a According to the factors, genotype (G), results of this study is presented. It also indicates increase, indicates decrea

Discussion
Recently, we developed a batt and exploration, muscle strength, [33]. The results, in males, indicate tional impairment in the quantitati matched NTg counterparts with no muscle weakness was determinant indicates decreases, and m, month.

Discussion
Recently, we developed a battery of psychomotor tests that include gait, neophobia and exploration, muscle strength, motor learning, physical resistance, and frailty status [33]. The results, in males, indicated that 3xTg-AD mice exhibit a more significant functional impairment in the quantitative variables of gait and exploratory activity than age-matched NTg counterparts with normal aging. The presence of movement limitations and muscle weakness was determinant for the functional decline related to the stages of severity of the disease that worsened with age. In addition, we detected the presence of signs of physical frailty, which accompany the functional deterioration of these animals. The signs of sarcopenia were present in an advanced stage of AD [31,32]. Therefore, the present study was designed to investigate, for the first time, several aspects: (1) from a gender-medicine perspective, the impact of this functional impairment in 3xTg-AD females as compared to males; (2) the long-term effects of repeated test, either in longitudinal (the same set of animals at 12 and 16 months of age) or transversal (two different sets, pre-tested or naïve, at 16 months of age) designs, both in pathological and normal aging scenarios; (3) to include a phenotype of frailty and physical deterioration that may find a functional correlation with the biological status (HPA axis and sarcopenia), with nuances in male and female animals. The survival curves on the cohorts of 191 animals allowed us to record higher mortality in females, being the group of NTg females the one that presented the highest number of deaths between 8-12 months of age. Interestingly, only females under the longitudinal design survived and achieved 16 months of age, while the group of naïve NTg females perished before reaching that old age, suggesting that repeated testing might have some protective effects. These results agree with our previous reports in these colonies, where high mortality rates associated with increased frailty were reported in females, and NTg exhibited increased mortality from 12 months of age [42]. In the case of 3xTg-AD mice, females that reached old age were survivors who overcame the disease's advanced neuropathological stages and exhibited lower behavioural differences with their NTg counterparts except for cognitive AD-hallmarks [47]. We have also described that, in male 3xTg-AD mice, an increase of mortality rates is associated with impairment in the neuro-immuneendocrine system compared to their females counterparts or the NTg genotype [48][49][50]. Noteworthy, we have recently reported survival bias and crosstalk between chronological and behavioral age in an APPswe model, where age-and genotype-sensitivity tests defined behavioral signatures in middle-aged, old, and long-lived mice with normal and AD-associated aging [51]. Therefore, the present work provides further evidence on sex and genotype-dependent differences in life expectancy and supports the key role of frailty and compensatory mechanisms as previously reported by our and other laboratories using different models of AD [29,[49][50][51][52].

Frailty
In the present work, the frailty results showed genotype differences between males, with NTg being the ones with the highest score. Only 12 of the 30 MCFI parameters were included as the incidence of the other indicators was very low or null. Kane and Brown [29] reported that 3xTg-AD male mice have a higher frailty index (FI) than NTg mice and 3xTg-AD females, and it was associated with their higher mortality ratios. Their study also indicated an increase in the frailty associated with age. On the other hand, in the present work, functional correlations in males found that their corticosterone levels correlated with frailty score and phenotype scoring system, both measures of functional decline. These results could indicate less deficit accumulation or functional capacity at the time of measurement in 3xTg-AD mice [53]. Therefore, it is plausible that other factors contribute to the survival/mortality of animals, and a complex multifactorial scenario be specific for each sex and biological age/stage of disease. In addition, in female C57BL/6 mice, greater frailty from 17 months of age with higher mortality at 26 months has been recently described in contrast to the non-fragile mice that reached 29 months of life [54]. These data have made it possible to identify that the prevalence of frailty in female mice increases throughout life and accurately predicts mortality [54]. Additionally, the animals' bodyweight presented genotype differences that coincide with previous data [33] but the re-test decreased the weight in males, probably due to the training carried out at 12 months of age.

Kyphosis
On the other hand, the severity of kyphosis was differentiated into postural and structural [31,32]. Here, genotype differences between males have been detected that corroborate previous reports, with greater severity in 3xTg-AD mice [31]. In females, here described for the first time, the severity of kyphosis increased with age and was more significant in the 3xTg-AD mice at 16 months in the re-test group, where the structural type predominates. The differences detected in males corroborate our other recent reports [32].

Phenotype Scoring System
Kyphosis is also one of the scores included in the phenotype scoring system [39,40], which has recently been functionally differentiated by a severity classification that allows more information to be collected in contrast to other variables, such as those associated with gait and exploratory activity [32]. Thus, in the phenotype scoring system, we detected that kyphosis at 12 months of age was more significant in NTg of both sexes, a significance that was not reproduced at 16 months in these animals, which corroborates our differentiation of severity in the presence of kyphosis since the postural condition can be positionally modified. In addition, in the gait score, an increase in functional impairment was detected in 3xTg-AD males and females, which appears in the re-test group at 16 months. This variable makes it possible to discriminate a significant impairment of movement and exploratory activity since bizarre behaviours may occur that interfere with movement [31]. The deficits detected in the quantitative parameters of gait will be discussed in the following section.

Clasping
Finally, the presence of increased clasping in naïve 3xTg-AD mice at 16 months can also be highlighted. It was related to a more significant involved or progression of the disease [55,56]. The present results also suggest that repeated tests exerted protective effects in this respect. Lalonde [55] described brain regions and genes affecting limbclasping responses. In the C57BL/6 strain, age-dependent locomotor deficits, including hindlimb clasping, are associated with a decreased number of dopaminergic neurons in aged mice, with reduced dopamine levels in the striatum [57]. Interestingly, alterations in the dopaminergic system described in 3xTg-AD mice and other AD models may also explain the presence of increased clasping.

Stride Length
Quantitative parameters in the gait analysis indicated that stride length was shorter in re-tested (16-month-old) male mice compared to age-matched naïve animals, and that this variable correlated with the gait phenotype score system. Interestingly, re-tested 3xTg-AD mice had the shortest stride length among the males compared to the naïve. In addition, differences in genotype and sex were observed at 12 and 16 months in the re-test group with greater stride length at 12 months in 3xTg-AD females and re-test in NTg males. In addition, the stride variability in females was lower than that of males, and the 3xTg-AD in all groups had the best performance, so their movement had more homogeneous steps throughout the trajectory. Previously, in our study in male 3xTg-AD mice of 6, 12, and 16 months of age, no differences in stride length or variability were detected, although a trend to increase stride length with age was observed in the case of 3xTg-AD mice while remained stable in the NTg genotype [31]. However, in another study at 6 months of age, increased stride length was reported in 3xTg-AD mice with no sex difference [58]. In addition, at 16 months of age, the gait of 3xTg-AD has been described as normal, without differences in genotype and sex [59,60]. According to the results, we propose that using the variability of the stride can help discriminate the trajectory of the movement during the gait analysis similar to humans where recently the variability was identified as a marker of cortical-cognitive dysfunction in AD patients [61,62].

Speed
A significant decrease in speed in the male 3xTg-AD mice in all groups was observed. This decrease may be associated with a progressive functional decline in the 3xTg-AD male mice and coincides with the findings at 13 months of age we have previously reported [33]. Cadence had a lower performance in the 3xTg-AD males at 12 months of age. However, it increased at 16 months in the re-test group, differing from naïve at this age. Thus, cadence and speed are the variables with the highest sensitivity to discriminate genotypic differences in male mice and differentiate changes in gait attributable to pathological aging in the 3xTg-AD genotype. In the case of 3xTg-AD females, speed in-creases slightly in the 16-months re-test group and was higher than in males in all groups. At the clinical level, the identification of early changes in gait is of great relevance for identifying psychomotor disorders that in the case of AD may be related to the timing of steps and gait speed [63]. Additionally, corticosterone levels were positively correlated with a cadence in males.

Neophobia and Exploration
The neophobia response, expressed as freezing, of 12 and 16-month-old naïve male mice was lower than in re-test mice in both genotypes, and statistically significant when contrasted with 16-month-old naïve mice. In females at 16 months of age, re-tested and naïve, a higher freezing was observed than in 16-month-old naïve females, albeit did not reach the statistical significance.
This neophobia emotional response is a characteristic of the 3xTg-AD model that is ac-companied by reduced immediate exploratory behaviour in a novel environment, as we first described in these animals in the open field test and the corner test already at the early 'premorbid' age of 2.5 months and worsened with the progress of the disease [64]. In addition, it corresponds to more sensitive ethological behaviours of the 3xTg-AD phenotype that has been reported in several other studies [31,33,42]. In addition, the horizontal exploratory activity did not report statistically significant differences.
However, in the vertical exploratory activity (number and latency of rearings), differences between the re-test male mice at 16 months and the naïve of the same age were more statistically significant than the activity in naïve mice. In addition, the ratio (visited corners/rearings) in the re-test male mice of 12 and 16 months increased in the re-test but differed from the females at both ages, being lower in males at 12 months in both genotypes. At 16 months in re-test, NTg male's ratio was high than NTg females, and in 3xTg-AD case, the ratio was increased in females 3xTg-AD. This decrease in activity over time, which is also observed in NTg mice, has been previously described as due to normal aging [64], with 3xTg-AD mice exhibiting less activity in most cases, which is attributed as a pathological trigger similar to BPSD that appear later in NTg mice due to normal aging [64]. In addition, in males was observed that correlated horizontal activity with triceps sural weight. Muscular strength is associated with global cognitive function in older people [65]. In addition, skeletal muscle mass index and physical performance (timed up and go test and grip strength) have decreased in older adults with AD [66]. Our results have not detected significant differences, although, at 12 months, it seems that females have a superior performance in grip strength and muscular endurance. Previously, we have reported that 13-month-old 3xTg-AD mice in natural isolation have preserved muscular strength [33] and that muscle strength and endurance would be associated with aging [31]. The laboratory of Brown also reported that at 6 months, 3xTg-AD mice have a deficit in grip strength [58], but at 16 months these results are not reproduced [59]. Additionally, the reduction in muscle weight and the appearance of sarcopenia may not yet be evident in the loss of muscle strength and resistance, or aging in this variable has greater importance than the distinction of the effects of the pathology in humans [67][68][69].

Geotaxis
On the other hand, geotaxis showed differences between the males, with the 3xTg-AD re-test at 16 months being the ones that obtained a worse performance and the 3xTg-AD naïve females at 16 months. In addition, females take longer to pass the test, which is reflected in the differences in GxS in the 16-month-old re-test and naïve group. The usefulness of this test has been previously described [70]. Specifically, the geotaxis has allowed us to differentiate the animals' postural positioning and balance strategies to pass the test and thus detect a possible functional deficit [31,33]. Therefore, 3xTg-AD re-test males and naïve females at 16 months show the most significant deterioration in this task.

Motor Performance: Learning and Physical Endurance-Rotarod
The motor performance showed superior performance in females of both genotypes. The motor learning tests and the number of trials reached the maximum values of the test in the re-test at 16 months. The increased performance may be due to pretraining done at 12 months, which can produce cognitive improvements with a long-term wheel of activity. In 16-month-old naïve 3xTg-AD males and females, lower latency and high number of trials were observed to achieve motor learning. Male 3xTg-AD mice have the most inferior performance in all tests.
As in motor learning, females have a high physical endurance. The 3xTg-AD females in the re-test group at 16 months achieved the highest performance over the male 3xTg-AD naïve and re-test, and female 3xTg-AD naïve 16-month-old females, and with similar performance to the NTg males of the same age. In addition, all groups increased their performance with training from Day 1 to Day 3, which is evident to a greater extent on the third day of training, and the effect of the re-test is observed at 16 months with an effect on different days for males and females, being in males on the first day of training and in females on the first and second day of training. Additionally, it was possible to distinguish the effect of aging in the naïve male NTg in contrast to the naïve at 12 months and re-test at 16 months. In addition, among the 3xTg-AD group, the sex differences between the 16-month-old re-test mice are distinguished from Day 1 to Day 3 of training. The 3xTg-AD males present the lowest performance among all groups, although with the training in the first day increased de physical endurance at 16 months in re-test group, on the following days, their performance is below 3xTg-AD naïve for 16 months.
The motor performance of 3xTg-AD mice has been reported in different studies. The performance in coordination and motor learning of 3xTg-AD mice has been highlighted over the performance of NTg mice, and these results are observable from 6 months and are reproduced at 16 months [58][59][60]. It has even been mentioned that 3xTg-AD females perform better than males at these ages [58,59]. In our laboratory, only reproduced the results of Stover et al. and Garvock-de Montbrun et. al. at 13 months, where the 3xTg-AD male mice presented a higher performance than the NTg, but in the latter, the weight factor interfered in the results [33]. Decreased motor function is also associated with aging, as reported in C57BL/6 mice of different ages [71][72][73]. In addition, we have differentiated the conceptualization of motor performance into motor learning-latency and motor learningtrials learning, since after physical exercise, the animals must manage to stay on a moving wheel in a coordinated manner. Consequently, in the first trials, physical endurance has a workload associated with an anaerobic exercise that progresses to aerobic exercise as the trials and their respective recovery times are replicated. In humans, the decrease in endurance exercise performance and its physiological determinants with aging appear to be mediated mainly by a reduction in the intensity (speed) and volume of exercise performed during training sessions [74]. Under this hypothesis, in their study, Pena et al. reported that 3xTg-AD mice improve their maximum latency in rotarod when subjected to aerobic exercise [75].
These results are accompanied by correlations with corticosterone levels and behave differently between males and females. In the case of males, corticosterone correlates positively with physical endurance on the first day of training, and in the case of females, it correlates positively with total physical endurance and physical endurance on the second and third days. On the other hand, a positive correlation was also detected in males between index-quadriceps sarcopenia and rotarod performance on the first and second days. A negative correlation was also detected between total physical endurance and the phenotype score system in females. Therefore, these interactions could explain the differences in performance between the groups studied. Corticosterone levels differed between groups due to sex and re-test factors, but not genotype. Males exhibited lower corticosterone levels in naïve mice of both genotypes, with similar levels between 3xTg-AD and NTg in the re-test group. On the contrary, in females, higher plasma corticosterone levels were observed in the 3xTg-AD re-test, and naïve females had similar levels that exceed the NTg re-test. It is also possible to distinguish that naïve 3xTg-AD females had higher levels than their male counterparts. The results agree with the sexual dimorphism reported by Muntsant et al., with higher plasma corticosterone levels in females [42], and also with plasma levels similar to the intervals described by Giménez-Llort et al. [76]. Additionally, corticosterone levels showed functional correlations with different variables depending on sex. In males, the correlation was inversely associated with the muscle mass of the quadriceps and triceps surae, and positively with frailty and gait cadence indicators. On the other hand, higher corticosterone levels correlated with higher performance on the first day of training in physical endurance. In females, the correlation with corticosterone was related to physical endurance performance with greater significance on the second and third training days. These results could indicate chronic stress if there is a long-term activation of the HPA axis in the case of females [77]. A report suggested that the combination of emotional and physical stress in a period of 5 h of exposure severely affected memory in NTg mice and increased the alterations in 3xTg-AD mice as a consequence of the reduction in the number dendritic spines and increase in the Aβ levels [50]. Additionally, the elevated corticosterone may precede cognitive impairments in genetically vulnerable 3xTg-AD females [78,79] and may, in turn, be related to frailty [80].

Sarcopenia
Furthermore, we have observed that the quadriceps and triceps surae muscles have a greater weight in naïve male mice, whereas in 3xTg-AD females, a lower weight is observed in the triceps surae muscle with significant differences with the group of 3xTg-AD females and re-test and males of this genotype. These differences in naïve 3xTg-AD females are also observed in the sarcopenia index of the triceps surae muscle. In humans, sarcopenia is closely related to dementia, particularly AD, and may be involved in the pathophysiological process of AD [68,81]. On the other hand, poor muscle function but not reduced lean muscle mass drives the association of sarcopenia with cognitive decline in old age [67,82]. Sarcopenia, low grip strength, and slow walking speed were significantly associated with mild cognitive impairment in the community-dwelling elderly [80,83]. Therefore, our results can be helpful to study what occurs in human pathology through a translational approach to motor dysfunction at different levels of disability [31].
Moreover, in the case of males, the weight of the quadriceps and triceps surae muscles negatively correlated with plasma corticosterone levels. A positive correlation of the quadriceps sarcopenia index with physical endurance Days 1 and 2 was also found. In the case of the sural triceps sarcopenia index, it correlated negatively with the number of corners visited in the exploratory activity. These correlations were not found in females.
Finally, the study's limitations were given by the high mortality rate of NTg females that resulted in the lack of 16-month-old naïve group. Therefore, the genotype differences between 3xTg-AD and NTg females could not be contrasted. However, the analyses were carried out to detect the sex differences between the 3xTg-AD group. In future research, it would be interesting to compare the results of this study with NTg females since their functional profile may differ from males in physical or biological variables, such as in 3xTg-AD females.

Conclusions
From the results, it is possible to highlight that the high mortality rate in females, and among them that in the NTg group, was prevented in the group of females behaviorally assessed at 12 months of age, and these females were able to reach the age of 16 months completing the longitudinal design. In addition, higher corticosterone levels were detected in females and lower muscle weight of the triceps surae, which could indicate sarcopenia and alteration of the HPAaxis, which was more significant in the naive group at 16 months. Additionally, there were genotype-sensitive variables such as the phenotype scoring system, frailty and kyphosis in which the group of 3xTg-AD males showed physical deterioration. In turn, the motor learning and physical endurance variables were sensitive to re-testing, with 3xTg-AD females achieving the best performance when repeating the behavioral battery at 16 months. In addition, the females exhibited a better performance in gait, where their stride was homogeneous and straight. Additionally, females exhibited less severe scores in physical variables, such as kyphosis, which could explain males' more significant deterioration in some motor tests. On the other hand, males showed deterioration in most of the variables studied. For their part, the correlations could explain the differences obtained between males and females, being positive in females between corticosterone and physical endurance, and the case of males between sarcopenia index and physical endurance as well as corticosterone with physical variables. The present results highlight the complexity of experimental scenarios in neurodegenerative diseases, such as Alzheimer's disease, confirming not only the different impact of factors depending on genotype, sex, and age but their interplay with the methodological approach. They provide evidence that genotype, sex and age-dependent impact of behavioral assessment, as well as the repetition of behavioral tests, should not be underestimated. Conversely, and most importantly, the ability of behavioral assessment and repeated tests to modify the behavioral outputs indicates that they could be considered functional trainings that modify survival, anxiety, and functional profile (physical endurance and motor learning) of old male and female 3xTg-AD mice and also NTg mice counterparts with normal aging.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/biomedicines10050973/s1, Table S1: Physical performance in males and females Naïve 12-month-old 3xTg-AD and NTg mice; Table S2: Physical performance in males and female 3xTg-AD and NTg after Re-test to 16m; Table S3: Physical performance in males and females Naïve 16-month-old 3xTg-AD and NTg mice; Table S4: Statistics Figure 5C.